The present invention generally relates to animal models useful for screening anti-adenovirus agents. Human adenoviruses (“Ads”) are a significant problem in immunosuppressed humans, especially in children undergoing allogeneic stem cell transplants. About twenty-percent of these pediatric patients develop disseminated Ad infections, and about half of these patients die. Unfortunately, there are no anti-viral drugs approved to treat these Ad infections. Cidofovir and ribavirin are used in some cases, but it is not known whether they are effective because they have not been studied in a systematic controlled manner. Anti-Ad drugs have not been studied in vivo because there is no animal model for replicating human Ads. This is because Ads have been considered to be highly species-specific. Therefore, it would be beneficial to provide a suitable animal model of systemic disease.
Ads have an icosahedral protein capsid that encloses a linear duplex DNA genome of 36 kpb and about 34 genes. Ads enter cells via receptor-mediated endocytosis and express their genes in the cell nucleus. Genes are expressed in two phases (1) an “early” phase, which precedes viral DNA replication, and (2) a “late” phase, which follows the initiation of viral DNA replication. Early gene products convert the cell into a factory for viral DNA replication, and late gene products are primarily components of the virion. Virus assembles in the cell nucleus by about one day post infection, and after about 2-3 days the cells begin to lyse and release infectious virus particles.
Human Ads are divided into six species, A, B, C, D, E, and F. There are 51 known human serotypes in the six species of human Ads: Species A (Ad12, 18, 31), Species B (Ad3, 7, 11, 14, 16, 34, 35, 50), Species C (Ad1, 2, 5, 6), Species D (Ad8-10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51), Species E (Ad4), and Species F (Ad40 and 41). Species B can be further divided into Species B1 (Ad3, 7, 11, 21, 50) and B2 (Ad11, 14, 34, 35). A serotype is defined based on neutralization with specific antisera.
The different Ads cause a wide variety of common and sporadic infections and there is not a strict one-to-one relationship between the serotype and disease (see Table 1 below). However, clear generalizations can be made. Ads in Species C, B1, and E cause respiratory tract infections, with Species C and the lower B1 serotypes (Ad3 and Ad7) predominating in infants and young children and Ad7 and Ad4 (Species E) causing acute respiratory disease (ARD) in military recruits. Species C and Ad3 cause conjunctivitis and pharyngealconjunctival fever. Species F (Ad40 and Ad41) causes gasteroentitis, especially in children. Species B2 infects the kidney and urinary tract (causing hemorrhagic cystitis). Species D, especially Ad8, 19, and 37, cause epidemic keratoconjunctivitis (EKC) throughout the world. For the most part, the molecular basis for these differences in pathogenicity is not understood. Ads not only cause disease as described above and as illustrated in Table 1 herein, Ads are also a significant problem in immunocompromised patients, as described below.
Ads have been isolated from immunocompromised hosts, in particular transplant patients, and have contributed to their morbidity and mortality. Pediatric allogeneic stem cell transplant patients under sever immunosuppression are particularly at risk. Roughly twenty-percent of these patients develop disseminated adenoviremia, usually of Species C serotypes but also of other serotypes as well.
The key factor associated with Ad disease is low T cell counts at the initial time of Ad detection in the blood. These low T cell counts are often the result of T cell depletion using agents such as alemtuzumab (a monoclonal antibody against CD52) in vitro and in vivo, selection of CD4+ stem cells in vitro, and continued immunosuppression following the transplant. The virus often appears in the blood about three weeks before the onset of symptoms, and virus DNA levels in the blood of greater than 106 to 107 copies/ml pose an increased risk for fatal outcome. Fatalities due to Ad are caused by hepatitis, pneumonia, and enteritis. Pediatric patients are more at risk for Ad, including disseminated disease and death, possibly because they are more commonly infected with the lower serotypes, especially Species C, and because their antibody and T cell response to Ad are not as developed as in adults who have species cross-reactive T cells. In pediatric transplant patients, the most common serotypes seen are in Species C, and these types can cause fatal disseminated disease. The use of real-time quantitative PCR (qPCR) is recommended to monitor Ad in the blood at successive times following transplantation, especially in T cell-depleted grafts, looking for an increase in viral load and, if possible, withdrawal of immunosupression. Another situation in which Ad is linked to immunosuppression is acquired immunodeficiency syndrome (AIDS). Twelve-percent of patients with AIDS have been reported to have Species B Ads in their urine.
TABLE 1DiseaseIndividuals most at riskPrincipal serotypesAcute febrile pharyngitisInfants, young children1-3, 5-7Pharyngoconjunctival feverSchool-aged children3, 7, 14Acute respiratory diseaseMilitary recruits3, 4, 7, 14, 21PneumoniaInfants, young children1-3, 7PneumoniaMilitary recruits4, 7Epidemic keratoconjunctivitisAny age group8, 11, 19, 37Pertussis-like syndromeInfants, young children5Acute hemorrhagic cystitisYoung children11, 21GastroenteritisInfants, young children40, 41MeningoencephalitisChildren and immunocompromised hosts7, 12, 32HepatitisInfants and children with liver transplants1, 2, 5MyocarditisChildren?Persistence:-Bone marrow transplant recipients;patients with acquiredimmunodeficiency or otherimmunosuppression syndromesIn urinary tract34-35In colon42-49Modified from refs. (164, 259), with permission.
With the emerging appreciation that Ads are a serious problem in immunosuppressed patients, there is strong interest in developing anti-Ad drugs. A class of drugs known as acyclic nucleoside phosphonates is effective against many viruses including Ads. One member of this class, (S)-9-[3-hydroxy-2-(phosphonomethyloxy)propyl] cytosine, known as cidofovir, has been studied extensively in Ad infections. Cidofovir is an analogue of 2′,3′-dideoxycytosine. Cidofovir is a monophosphate, and it is converted to the di- and triphosphate forms by cellular enzymes. These compounds have much higher affinity for viral DNA polymerases than for cellular DNA polymerases, thereby providing specificity for virus-infected cells. They act as inhibitors of the polymerase, and the triphosphate is a substrate for the polymerase, acting as a DNA synthesis chain terminators. Cidifovir is approved for treatment via the intravenous route for cytomegalovirus retinitis in AIDS patients.
Cidofovir is a potent, nontoxic inhibitor of Ad replication in cell culture, including serotypes 1, 2, 5, and 8. As shown in FIG. 1, cidofovir inhibits the replication of our Ad5-based vector named VRX-007. In a recent study, cidofovir inhibited replication in HEp-2 cells of serotypes from Species A, B, D, E, and F. Only Species C was inhibited by ribavirin. A549 cells were mock-infected or infected with 10 or 0.1 plaque-forming units (PFU)/cell of VRX-007. Concomitantly, cidofovir was added to the medium at the indicated concentrations. The cells were stained with crystal violet dye at seven days post infection. Cidofovir has shown some efficacy in the rabbit and cotton rat models of ocular models of Ad5 infection.
A large multi-center trial was initiated in the United States to evaluate cidofovir against Ad ocular infections in humans. Significant efficacy was observed, but the trial was discontinued because of a narrow efficacy/toxicity ratio. Two other known clinical trials have been conducted for cidofovir treatment of EKC. In the first trial, no efficacy was seen using 0.2% cidofovir plus 1% cyclosporine. In the second trial, 1% cidofovir plus 1% cyclosporine lowered the frequency of severe corneal opacities but caused local toxicity.
Cidofovir has also been examined in a number of retrospective studies as well as in case reports, alone or sometimes in combination with ribavirin, to treat Ad in immunosuppressed patients. However, there have not been controlled clinical trials for systemic use of these drugs to treat Ad, and the drugs have not been licensed for this use. Cidofovir showed some efficacy against Ad in patients immunosuppressed because of stem cell and bone marrow transplants. Cidofovir was effective against Ad-associated hemorrhagic cystitis and renal dysfunction in bone marrow transplant patients. In a known prospective study of eight pediatric stem cell transplant patients, cidofovir treatment seemed to provide long term suppression of Ad without dose-limiting nephrotoxicity. In a recent known prospective study in which Ad was detected in 26 of 155 pediatric stem cell transplant patients, ribavirin was used when Ad was first detected and cidofovir was used in patients with persistent viremia. Although not curative, the antiviral therapy appeared to control the Ad infection. Cidofovir seemed to resolve disseminated Ad in a pediatric liver transplant patient and Ad7 in a B cell lymphoma adult patient with meningoencepahlitis. Although these various studies are somewhat encouraging, one major problem with the systemic use of cidofovir is nephrotoxicity caused by accumulation of the drug in renal proximal tubules.
Because of the presence of the phosphonate group on cidofovir, the drug shows poor oral bioavailability. However, a new series of bioavailable ether lipid-ester prodrugs of cidofovir and a related acyclic nucleoside phosphonate has been developed that were reported to be 15-2,500-fold more effective in inhibiting Ad in cell culture.
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